Polymeric fibre and method for making same

ABSTRACT

A method of producing a polymeric fibre comprising dissolving at least one fibre forming polymer in a solvent so as to form a polymer solution, and feeding the polymer solution under gravity through an orifice directly into a non-solvent whereby to cause formation of a polymeric fibre in the non-solvent. The method of producing a poly(ε-caprolactone) fibre comprising dissolving poly(ε-caprolactone) polymer in a solvent whereby to form a poly(ε-caprolactone) solution, and feeding the poly(ε-caprolactone) solution through an orifice directly into a non-solvent whereby to form said poly(ε-caprolactone) fibre.

This invention relates to a novel method of producing a polymeric fibre,particularly but not exclusively, a poly(ε-caprolactone) (PCL) fibre,for use in the production of biomedical implants and cell-supportmatrices utilised in tissue engineering, or in biodegradable textiles.

Tissue engineering is currently attracting great interest because of theprospects for obtaining ‘living’ tissue replacements and therebyreducing the reliance on donor tissue and organs. Tissue engineeringinvolves the design and manufacture of implants for repair, support,augmentation or replacement of damaged or diseased tissues and organssuch as bone and skin. In one approach, cells are seeded onto 3-Dscaffolds or matrices ex-vivo. The cell-scaffold construct issubsequently implanted where cell development continues to regeneratenew tissue. The microstructure and architecture of the scaffold,together with the surface chemistry exert profound effects on celldistribution, morphology and alignment and, importantly, cellproliferation and differentiation which underpins correct tissuedevelopment (Freed, L. E. and Novakovic, G. Culture of organised cellcommunities, Adv. Drug. Del. Rev. 33 (1998) 15-30. Hubbell, J. A.Biomaterials in tissue engineering, Biotechnology, 13 (1995) 565.Marler, J. J. Transplantation of cells in matrices for tissueregeneration, Adv. Drug Del. Rev. 33 (1998) 165-182).

The scaffold component of tissue engineered constructs has been producedusing a variety of techniques including fibre bonding (Mikos, A. G etal, Preparation of PGA bonded fibre structure for cell attachment andtransplantation, J. Biomed. Mater. Res. 27 (1993) 183), solid free-formfabrication (Koegler et al, Solid free-form fabrication of boneregeneration devices, 1st Smith and Nephew International Conference,York, July 1997) and salt extraction (de Groot, J. H et al, Use ofporous PU for meniscal reconstruction and meniscal prostheses,Biomaterials 17 (1996) 163-173). In particular, inherently porous, fibreconstructs of various designs based on woven, knitted and non-woventechnologies have been widely investigated for improving cell attachmentand tissue infiltration of the scaffold. (Wintermantel, E. et al, Tissueengineering scaffolds using superstructures, Biomaterials 17 (1996)83-91). The design and production advantages associated with polymerfibres have already led to the use of both natural and synthetic fibresfor a wide range of tissue repair applications involving bone andcartilage, skin substitutes, nerve regeneration (Marler, J. J. supra)and blood vessels (Hanson, S. J, Mechanical evaluation of resorbablecopolymers for end use as vascular grafts, ASAIO Trans. 34 (1998)789-93). Indeed the early observations of favourable cell growth on silkfibres stimulated much of the research into biomaterials-cellinteraction. Fibrous mats or meshes of synthetic resorbable polymerssuch as polyglycolic acid (PGA) or poly(DL lactide co-glycolide) (PLG)are being investigated extensively as scaffold materials for seededcells in tissue engineering (Freed, L. E and Novakovic, G. supra,Hubbell, J. A. supra, Marler, J. J. supra). One production techniqueentails heating of a wad of PGA fibres in a mould to produce pointwelding between the individual fibres (Mikos, A. G et al supra). Oncooling the mesh retains the shape of the mould and is used as atemplate to support cell adhesion and tissue infiltration.

Resorbable synthetic fibres such as ‘Dexon’ (produced from PGA) andpolyglactin have been used for many years as suture materials.Resorbable sutures are designed to maintain wound closure for fairlyshort times of around 6 weeks to coincide with the relatively rapidrepair processes in skin and soft tissues. Thus the resorption rate ofthe suture is correspondingly rapid and the mechanical propertiesdecline over approximately 2 weeks to promote natural strengthening ofthe repair site. The high resorption rate of PGA and some PLG copolymersof 4-6 weeks renders them highly suitable for application as sutures andscaffolds for soft tissue repair but presents problems in applicationssuch as bone repair which require longer times for tissue growth (about6 weeks) and remodelling (about 6 weeks). Fast-resorbing polymers suchas PGA and PLG also display high shrinkage values (Coombes, A. G. A. andMeikle, M, Resorbable synthetic polymers as replacements for bone graft,Clinical Materials 17 (1994) 35-67) which can mean thatanchorage-dependent cells and tissues are not presented with a stablesubstrate for laying down extra-cellular matrix (ECM). In addition, theproduction of acidic degradation species by fast-resorbing polymers cancompromise tissue repair (Kyriacos, A et al, Sterilisation, toxicity,biocompatibility and clinical, applications of PLA/PGA copolymers,Biomaterials 17 (1996) 93-102).

Slow-resorbing fibres based on polylactic acid (PLA), having resorptiontimes in excess of one year, provide extra scope for producing implantsmatched to tissue repair rates and characteristics. The production ofPLA fibres by melt spinning and solution spinning (see below) has beendescribed (Eling, B, Biodegradable materials of poly(L-lactic acid) 1,Polymer 23 (1982) 1587-93. Leenslag, J. W et al, Resorbable materials ofpoly(L-lactide) V Influence of secondary structure on the mechanicalproperties and hydrolysability of poly(L-lactide) fibres produced by adry spinning method, J. App. Polym. Sci. 29 (1984) 2829-2842. Fambri, L.et al, Biodegradable fibres. Poly-L-lactic acid fibres produced bysolution spinning, J. Mater. Sci. Mater. in Medicine. 5 (1994) 679-683)and the mechanical, thermal and morphological properties have beendetailed.

Fibre Spinning

Fibre spinning may be broadly divided into two categories, melt spinningand solution spinning. In melt spinning, the molten polymer is forcedthrough a spinneret and the jet of molten polymer is cooled to formsolid threads. These threads are subjected to a drawing procedure tocontrol chain orientation and fibre tensile properties. Solutionspinning is based upon extrusion under pressure of concentrated polymersolutions. In dry solution spinning, the extruded filaments are dried toremove solvent, whereas in wet solution spinning the filaments areextruded into a non-solvent to precipitate the polymer in the form of athread. Solution spinning of the poly(α-hydroxy acids) such as PLAnormally requires high solution viscosities to enable extrusion of afilament, prior to drawing. High strength polyethylene (PE) fibres havebeen spun from solutions undergoing shear flow in a Couette apparatusrather than by extrusion through an orifice. Investigations demonstratedthe marked influence of spinning temperature on fibre strength andstiffness occasioned by the improvement in polymer chain alignment andreduction of the chain folded element (‘kebab structure’) in the fibrous‘shishkebab’ structures which made up the fibre. In another approach,high modulus, high strength fibres have been produced by controlleddrawing of PE gels (Smith, P et al, Polymer Bulletin. 1 (1979) 733) orpoly(vinyl alcohol) gels (Cebe, P and Grubb, D, Gel drawn fibres ofpoly(vinyl alcohol), J: Mater. Sci. 20 (1985) 4465-4478).

Fibres for Drug Delivers

Hollow fibres have been produced as reservoirs for drugs to achievecontrolled delivery on implantation (Schakenraad, J. M et al,Biodegradable hollow fibres for the controlled release of drugs,Biomaterials, 9 (1988) 116-120). Hollow PLA fibres were spun using a‘dry-wet’ coagulation spinning process using a 15% PLA, 80% dioxane, 5%polyvinylpyrrolidone solution at 50° C. Water was used as the internaland external coagulant. The hollow fibres (0.72 mm OD, 0.47 mm ID) weresubsequently filled with levonorgestrel in castor oil (25% w/w) and heatsealed to provide lcm long, injectable devices.

Potential Advantages of PCL Fibres

PCL fibres would provide a useful, low cost, alternative to PLA. PCL isnoted for its biocompatibility (Pachence, J. M., Kohn, J., BiodegradablePolymers in Principles of Tissue Engineering pp263-277, Eds. Lanza, R.P., Langer, R., Vacanti J., Academic Press 2000, 2^(nd) Edition) andlike PLA, PCL is a slowly degrading poly(α-hydroxy acid) which wouldallow more time for regenerating tissue to establish. PCL (in mouldedform) exhibits lower tensile modulus (0.3 GPa) and strength (19 MPa)than PLA (4 GPa, 70 MPa) but higher extensibility which presentsopportunities for adjusting the compliance of cardiovascular implantsfor example. PCL films have been shown to provide a favourable substratefor growth of bone cells (Ali, S. A et al, Mechanisms of polymerdegradation in implantable devices 1. PCL, Biomaterials 14 (1993)648-656) and the polymer has been applied as the matrix component incomposites intended for bone repair (Marra, K. G et al, In vitroanalysis of biodegradable polymer/hydroxyapatite composites for bonetissue engineering, J. Biomed. Mater. Res. 47 (1999) 324-35).

PCL fibres have been produced by melt spinning at 210° C. followed bydrawing at 45° C. to give draw ratios ranging from 5-9 (Mochizuki, M etal, Hydrolysis of PCL fibres by lipase, J. App. Pol. Sci. 55 (1995)289). Tetracycline-containing PCL fibres have been investigated forperiodontal therapy for example (Goodson, J. M et al, Monolithictetracycline-containing fibers for controlled delivery to periodontalpockets, J. Periodontol. 54 (10) (1983) 575-9) and mention has been madeof 0/60/120° patterns of PCL fibres forming scaffolds for tissueengineering (Hutmacher, D. W, Tissue engineering research. MedicalDevice Technology, 1 (2000) 33). These studies both featured melt spunfibres.

It is an object of the present invention in one aspect to provide anovel method of producing a wet spun polymeric fibre.

According to a first aspect of the present invention there is provided amethod of producing a polymeric fibre comprising:

-   -   (i) dissolving at least one fibre forming polymer in a solvent        so as to form a polymer solution, and    -   (ii) feeding the polymer solution under gravity through an        orifice directly into a non-solvent whereby to cause formation        of a polymeric fibre in the non-solvent.

It will be understood that the viscosity of the polymer solution must besufficiently low to allow the solution to pass through the orifice undergravity. This may be achieved by having a relatively low concentrationof the at least one polymer in the solution, or through the inherentlylow viscosity characteristics of the at least one polymer.

It will be further understood that the choice of solvent/non-solventwill depend upon the particular polymer(s) being used. Preferably, thenon-solvent is chosen such that the polymer solution is denser than thenon-solvent, thereby allowing free flow of the polymer solution streamand avoiding floatation of the polymer solution on the non-solvent.

As used herein, the term “non-solvent” includes liquids in which thepolymer is sparingly soluble. The key functional requirement is that thepolymer is sufficiently less soluble in the non-solvent to induce fibreformation therein. Thus the term “non-solvent” includes within its scopemixtures of solvents and non-solvents which have sufficiently lowsolubility for fibre formation.

Preferably, said at least one fibre-forming polymer is selected from alinear aliphatic polyester e.g. poly(ε-caprolactone), a polylactide, apolyglycolide, their copolymers with (i) other aliphatic polyesters,e.g. poly(DL lactide co-glycolide), poly(glycolide ε-caprolactone), and(ii) monomers other than linear aliphatic esters e.g. poly(glycolidetrimethylene carbonate), poly(L-lactic acid-L-lysine),poly(DL-lactide-urethane), PLA/PEO copolymers, poly(ester-amide), apolyanhydride, a polyorthoester, a poly(ester-ether) e.g.poly-p-dioxanone, a polyphosphazine, PHB, PHV and their copolymers, apoly(β-malic acid), a poly(amino acid) e.g. poly(L-lysine), an aliphaticor an aromatic polycarbonate e.g. poly(ethylene carbonate) and theircopolymers with any of the aforementioned polymers.

Blending of polymers to optimise material properties has been appliedextensively in biomedical materials and drug delivery research. Themethod of the invention is potentially useful for blending one or moredifferent polymers (synthetic or-natural) to obtain differentphysico-chemical characteristics so as to control the pattern of drugrelease from the fibre or to modulate cell interaction. It is known thatthe degradation rate of the poly(α-hydroxy acids) such as PLG, forexample, can be varied from several weeks to over a year bycopolymerisation, control of molecular weight, crystallinity andmorphology. Blending of such polymers would allow control of thedegradation characteristics of the product fibres.

It will be understood that the choice of polymers to be blended willdepend upon the desired characteristics of the formed fibre. Thus, step(i) may include the addition of at least one additional polymer. Usefulsynthetic polymers include copolymers produced from lactide andnon-lactide monomers such as lactones (e.g. epsilon caprolactone) orethylene glycol, PMMA, PU, copolymers containing a thermoplasticelastomer or hydrogel forming copolymers such as poly(hydroxyethylmethacrylate).

Further useful polymers include polyethylene glycol (PEG), polyethyleneoxide (PEO) copolymers of poly(ethyleneoxide)-poly(propylene oxide)(Pluronic, Tetronic copolymers), polyvinylpyrrolidone (PVP) resulting infibres having a water soluble phase. Such compositions may be useful forcontrolling the rate and time of drug release from the polymer matrix.

Step (ii) may be effected by feeding the polymer solution through theorifice simultaneously with a pre-formed fibre, such that the resultantfibre comprises a core of the pre-formed fibre surrounded by a fibreformed from the polymer solution. The pre-formed fibre may be, forexample, polyester (e.g. Dacron (Trade name)).

Where the at least one fibre-forming polymer is poly(ε-caprolactone),said solvent is preferably selected from one or more of acetone, ethylacetate, dichloromethane, chloromethane and chloroform. Most preferably,said solvent is acetone.

Where the at least one fibre-forming polymer is poly(e-caprolactone)said non-solvent is preferably selected from one or more of methanol,ethanol and water. Most preferably, said non-solvent is methanol.

Preferably, said solvent and non-solvent are miscible.

Preferably, the method includes an additional step of introducing intothe solvent one or more additives prior to step (ii).

Said additional step may be effected prior to, concomitantly with orafter step (i). The or each additive may be chosen so as to vary one ormore properties of the fibre such as degradation rate, density, thermal,mechanical, morphological chemical characteristics and cell growthproperties. The or each additive may be solid, particulate or fibrousand substantially insoluble in the polymer and solvent, or the or eachadditive may be liquid and/or soluble in the polymer and/or solvent.

Preferably, said additional step is effected by low shear mixing.Preferably, after step (ii) said additive(s) is/are homogeneouslydistributed in the fibre.

The process of the present invention is effected under low shear and lowtemperature conditions. Thus, additives which are temperature sensitive(such as agents which may be deactivated, denatured or which decompose)and those which are deactivated under high shear may be incorporated insaid additional step. It has not previously been viable to incorporatesuch additives in melt spun (high temperature) or traditional solutionspun (high pressure/shear) processes. Examples of such additives includepeptides, proteins and DNA pharmaceuticals.

Examples of useful additives include natural materials such aspolysaccharides (e.g. inulin, starch, dextran, cellulose andderivatives), sugar spheres, extra-cellular matrix components such asglycosaminoglycans and proteins (e.g. collagen, gelatin and albumin).

Other useful additives include bioceramics such as hydroxyapatite (HA),carbonate hydroxyapatite, tricalcium phosphate (TCP), carbon, calciumcarbonate, and ‘Bioglass’ (a highly bioactive glass particulate materialused in bone repair).

Examples of useful synthetic polymer additives include PMMA powders suchas those used in bone cements for implant fixation, polyesters (PET),biodegradable polymers such as PLA and PLG, polyorthoesters,polyanhydrides and oligosaccharide ester derivatives (OEDs).

Examples of useful discontinuous fibrous additives include alumina,carbon and synthetic polymers such as polyester (‘Dacron’), PGA andpolydioxanone (PDS).

It is known that protein adsorption onto biomedical implants in vitroand in vivo directly influences initial cell attachment and adhesion andthe subsequent stages of cell spreading, proliferation anddifferentiation (Hubbell, J. A. supra). Survival of many celltypes-including fibroblasts and endothelial cells requires integrinmediated adhesion to extracellular matrix proteins. Modification of thesurface of polymeric fibres by cell-adhesion proteins such asfibronectin or peptides having cell binding properties provideadvantages for tissue engineering.

Thus, the additional step preferably includes the introduction of atleast one cell adhesion molecule for modifying the fibre surface (e.g.by adsorption). The cell adhesion molecule may be selected from:collagen, gelatin, fibronectin, vitronectin, laminin, elastin and theirsynthetic analogues such as the protein-silk polymers or conjugates ofsuch molecules with hydrophobic moieties, synthetic analogues ofbiomolecules containing cell binding sequences (such as the RGDsequence), antibodies having affinity for specific cell receptors ofinterest and molecules having affinity for cell surface polysaccharides.

The use of various growth factors has been investigated for regenerationof skin, bone, cartilage, nerves and blood vessels. However, there is aneed to control the time, extent and sequence of growth factor deliveryto responsive cells to mimic physiological processes and to maximise thetherapeutic potential of these agents. The controlled delivery andpresentation of polypeptide growth factors, or DNA which encodes forthese factors, by polymeric fibres would be advantageous for productionof tissue engineered constructs.

Thus, the additional method step preferably includes the introduction ofat least one additive selected from polypeptide growth factors such astransforming growth factor-β (TGF-β), VEGF, EGF, BMP, IGF, or DNAencoding for polypeptide growth factors, their synthetic analogues orconjugates with other molecules such as PEG.

Further useful additives include, vaccine antigens, therapeuticantibodies, DNA, RNA, oligonucleotides, anti-coagulants, anti-cancer,anti-inflammatory, anti-bacterial and anti-viral agents, thrombolyticagents, hormones and decalcified freeze-dried bone (DFDB). Other usefuladditives include bioactive compounds for veterinary and agriculturaluse (e.g. pesticides, plant nutrients and growth hormones).

It will be understood that incorporation of such molecules into the bodyof the fibre facilitates controlled release of said molecules, the rateof release being controllable by one or more other additives aspreviously described.

The or each additive may comprise one or more active agent and a carriervehicle. Examples of such carrier vehicles include but are not limitedto: sugar spheres or spheres produced from polysaccharides such asdextran, inulin, starch, cellulose and derivatives, proteins such asgelatin and albumin. Examples of suitable synthetic carrier particlesinclude synthetic non-resorbable polymeric particulates, microspheresand nanospheres (e.g. polyamide, PET, PMMA) and resorbable polymerparticulates, microspheres or nanospheres (e.g. PLA), carbon,bioceramics (e.g. hydroxyapatite) and magnetic particles. The activeagent(s) may be incorporated into the carrier vehicle or coatedthereupon.

The method may comprise a further additional step (iii) of applying atleast one additive to the surface of the fibre formed in step (ii).

Step (iii) may be in addition to or instead of the aforementionedadditional step and the or each additive used in step (iii) may beselected from any of those already referred to above.

According to a second aspect of the present invention there is provideda fibre producible by the method according to the first aspect.

According to a third aspect of the present invention there is provided amethod of producing a poly(ε-caprolactone) fibre comprising:

-   -   (i) dissolving poly(p-caprolactone) polymer in a solvent whereby        to form a poly(ε-caprolactone) solution, and    -   (ii) feeding the poly(ε-caprolactone) solution through an        orifice directly into a non-solvent whereby to form said        poly(F-caprolactone) fibre.

Preferably, said poly(ε-caprolactone) solution is passed through theorifice under gravity.

The method according to the third aspect may include either or both ofthe additional steps described in relation to the first aspect. It willtherefore be understood that any of the solvents, non-solvents andadditives mentioned in the context of the first aspect are alsoapplicable to the third aspect. However the solvent is preferablyacetone. The non-solvent is preferably methanol.

According to a fourth aspect of the present invention there is provideda poly(ε-caprolactone) fibre producible by the method according to thethird aspect.

Examples of fibres according to the present invention will now bedescribed, by way of example only, with reference to the accompanyingdrawings in which:

FIG. 1 is an electron micrograph showing surface topography of PCLmelt-spun fibres for comparison with the fibres of the presentinvention,

FIG. 2 is an electron micrograph showing the surface topography of anas-spun PCL fibre,

FIG. 3 is a plot of cell proliferation against time for PCL fibres madein accordance with the present invention containing progesterone versusTCP and PCL controls

FIGS. 4 to 6 are graphs of cell density plotted against time for-cellsgrown on PCL fibres and tissue culture plastic as control, and

FIG. 7 is an electron micrograph showing the surface topography of asurface modified PCL fibre.

METHODS

Unless otherwise stated, poly(ε-caprolactone). (PCL) fibres wereproduced from polymer of average molecular weight (Mw) 115,000 (Meanmolecular weight (MW) 50,000 based on measurement of reduced viscosity).

Fibre macroscopic shape and cross-sectional dimensions were determinedusing an optical microscope having a calibrated eyepiece graticule.Fibre morphology was examined using scanning electron microscopy (SEM).

Fibre tensile properties (Young Modulus (E-modulus), ultimate tensilestrength (UTS) and elongation at break) were measured using a HounsfieldH10KS tensile testing machine. Fibre extension was measured using 25 mmlength specimens and approximated from the crosshead movement. Testingwas performed at a crosshead speed of 15 mm/minute.

It is well known that secondary processing operations (fibre draw ratioand drawing temperature) influence fibre properties. The effect of colddrawing on fibre properties was evaluated by controlled extension offibre samples at room temperature at a rate of 15 mm/min using thetensile testing machine. The tensile properties of drawn fibres weresubsequently determined as described above.

The thermal characteristics of PCL fibres (melting point (Tm) andpercentage crystallinity) were determined by differential scanningcalorimetry (DSC) using a heating rate of 10° C./min. The latterproperty was estimated from area under the curve measurements using avalue of 139.5 J/gm for the heat of fusion of fully crystalline PCL(Pitt et al, Aliphatic polyesters. 1, The degradation ofpoly(ε-caprolactone) in vivo, J. App. Polym. Sci. 26 (1981) 3779-3787).Surface modification of PCL fibres with protein was achieved byadsorption of gelatin from 5, 10 and 20% w/v solutions respectively.Fibres (30 mg) were dip coated and dried overnight at room temperature.The coated fibres were washed by immersion in 70% ethanol then water forseveral minutes.

The amount of adsorbed gelatin on the fibres was determined directlyusing the BCA total protein assay. Coated fibres (30 mg) were immersedin bicinchonic acid protein (BCA) reagent (Sigma Chemicals) in the wellsof a 96-well plate and heated at 60° C. for 15 minutes. The absorbanceat 562 nm was read using a plate reader and the weight of protein wasestimated by comparison with a calibration curve.

Protein loading of fibres was achieved by suspension of powderedovalbumin (OVA, Sigma) in a 10% PCL solution to give a suspensionconcentration of 1% w/v. Fibre spinning was subsequently carried outusing the suspension.

Protein release characteristics were determined by incubating OVA-loadedfibres in phosphate buffered saline (PBS) at 37° C. and assaying therelease medium for protein content at day 1, 2 and 7 using the BCAassay.

The amount of protein exposed at the surface of OVA-loaded fibres afterincubation in PBS for 1, 2 and 7 days respectively, was determined bydirect assay of fibres as described above for gelatin-coated fibres.

Cell interaction with PCL fibres was assessed using cell culturemethods. Fibroblasts (Swiss 3T3 cells) and myoblasts (C9C12) at adensity of 40,000/ml, were each seeded onto PCL fibres contained in24-well plates. Tissue culture plastic (TCP) was used as a control. Thenumber of attached cells was counted at time periods up to 8 days. Ateach time point, non-adherent cells were removed by washing in PBS. PCLfibre samples and TCP with attached cells were subsequently treated withZaponin (Sigma Chemicals) to lyse the attached cells and the cell nucleiwere counted using a Coulter counter. Cell attachment was subsequentlyquantified in terms of cell number/fibre area.

Separate samples of PCL fibres with attached cells were dried using agraded series of ethanol/water mixtures and shadowed with gold prior toexamination of cell morphology using SEM.

Additionally, cell interaction and growth of human umbilical veinendothelial cells (HUVECs) on as-spun PCL fibres (150 μm fibres producedusing a 10% (w/v) solution of PCL in acetone) were investigated usingcell culture methods to assess the biocompatibility of PCL fibres andtheir potential as scaffold materials for soft tissue engineering.HUVECs were seeded at a density of 50,000/ml onto PCL fibres wrappedaround 22 mm×22 m glass coverslips contained in 6-well plates (Costar).Tissue culture plastic (TCP) was used as a control. The number ofattached cells was counted at 1, 2, 4, 7 and 9 days. At each time point,non-adherent cells were removed by washing the samples in HBSS (Gibco).PCL fibre samples and TCP with attached cells were subsequently treatedwith trypsin (Gibco) for 5 minutes to detach the cells and the cellnumber was counted using a haemocytometer (Imp. Neubauer. WeberScientific Int. Ltd). Cell attachment was subsequently quantified interms of cell number/fibre surface area, the fibre contact area beingestimated using 50% of the fibre circumference.

Production of PCL Fibres.

A pre-weighed amount of poly(ε-caprolactone) was dissolved in a selectedsolvent (e.g. acetone) and made up to a predetermined volume to give apolymer solution of a desired concentration. The polymer solution wasadded to a reservoir of a spinneret having an adjustable flow control.Conveniently the spinneret may be produced from glass having acylindrical barrel (reservoir section) (95 mm long×7 mm internaldiameter) tapering to a capillary (50 mm long×1 mm internal diameter).The free end of the capillary of the spinneret was positioned in a bathcontaining non-solvent (e.g. methanol) such that it was immersed in thenon-solvent. The polymer solution was allowed to flow under gravitythrough the capillary of the spinneret into the non-solvent bath so asto form a fibre. The ‘as spun’ fibre was taken up on a variable speedmandrel positioned above the non-solvent bath.

The method was varied as necessary to incorporate one or more additivesinto the polymer solution. If desired, the as-spun fibres weresubsequently drawn to modify fibre properties such as tensile strengthand morphology.

The method of the invention avoids pressurised flow conditions which candisrupt flow of the solution and produce fibre surface irregularities.This in turn can interfere with cell attachment and growth on the fibre.In addition the low shear conditions avoid shear-induced degradation ofbiopharmaceuticals such as polypeptide growth factors which may beincluded in the spinning solution.

Modification of fibre surface topography was achieved by wrapping wetas-spun PCL fibres around a mandrel submerged in the methanol bath priorto the fibre drying stage. The mandrel surface exhibited a machinedtopography characterised by a peak-to-peak separation of 91 μm and apeak height of 30 μm.

Comparative Examples

Production of Poly(α-Hydroxy Acid) Fibres.

Solution Spinning of Poly(α-Hydroxy Acid) Polymers.

Dry spun PLA fibres have been produced by extrusion of polymer solutionsin toluene at 110° C. through a conical capillary with diameter 1 mm,followed by drying at room temperature and hot drawing (Eling, B.supra). As-spun fibres were produced at rates between 0.25 and 0.35m/min. PLA fibres with molecular weight (Mw) below 3.5×10⁵ could not beobtained due to the low solution viscosity at 110° C. PLA fibres havealso been produced by dry spinning from solution in good solvents suchas dichloromethane at rates between 0.02 and 1 m/min, followed by hotdrawing (Gogolewski, S and Pennings, A. J, Resorbable materials ofpoly(L-lactide) II, Fibres spun from solutions of poly(L-lactide) ingood solvents, J. App. Pol. Sci. 28 (1983) 1045-1061). A similar dryspinning method for PLA fibres was described by Fambri et al supra whichinvolved extrusion of chloroform solutions through a needle of internaldiameter 1 mm and length 15 mm at rates between 0.01 and 2 m/min.As-spun fibres were subsequently hot drawn at temperatures between 150and 210° C. PLA fibres having a loosened fibrillar structure, toincrease degradation rate, have been produced by incorporation ofadditives such as camphor or polyester urethanes in the PLA solutionprior to dry spinning (Leenslag, J. W et al supra).

Melt Spinning

Melt spun PLA fibres have been produced at rates between 0.25 and 0.35m/min by extrusion of a polymer cylinder at 185° C. through a capillarywith diameter 1 mm and length 10 mm (Eling, B. supra)., Fibres could notbe produced at temperatures above 185° C. because of the low meltviscosity.

Properties of PLA Fibres

The tensile strength and morphology of PLA fibres dry spun from solutionwere found to be strongly dependent on the molecular weight andconcentration of PLA in the spinning solution and upon the drawratio/temperature. Fibres of PLA with high tensile strength (1.2 GPa)and Young modulus (12-15 GPa) have been produced by hot drawing solutionspun fibres (Eling et al, supra). At least one PLA fibre product(‘Lacton’ from Kanebo Goshen) is commercially available. However, PLAfibres suffer from low compliance (Leenslag et al, supra), a tendency todegrade during melt processing and the starting polymer is expensive.

Melt Spinning of PCL Fibres

FIG. 1 (Hutmacher et al., supra) shows the typical smooth surfacetopography of melt spun PCL fibres. Plate a) of FIG. 1 is a freezefracture cross-sectional surface and plate b) is a top view of a PCLscaffold with a 0/60/120° lay down pattern.

EXAMPLES

Fibre Processing/Properties Relationships.

The production rate of continuous PCL fibres under various processconditions was established by systematic variation of thesolvent/non-solvent system, PCL molecular weight and solutionconcentration. In the following experiments the solvent used wasacetone, and the non-solvent used was methanol unless otherwise stated.

PCL fibres were produced continuously at speeds typically ranging from0.8 to 2.5 m/minute. The maximum rate of fibre production was found tobe influenced by the nature of the solvent/non-solvent system, PCLmolecular weight and the solution concentration, the effect of solutionconcentration being shown in Table 1. Fibre production rate was higherat low solution concentration in line with the higher flow rate of thelower viscosity solutions. TABLE 1 Production rates of as-spun PCLfibres. PCL conc. Fibre production rate Fibre diameter (% w/v) (m/min)(μm) 5 fibres not formed 6 1.5-3.5 140-240 10 1.5-2.7 150 15 1.3-2.5 15020 0.9 150Fibre Dimensions and Morphology.

As-spun PCL fibres are roughly circular in cross-section and exhibit arough, porous surface (FIG. 2). This is in contrast to the smoothsurface exhibited by melt-spun PCL fibres (FIG. 1). Molecular weight ofthe polymer used affects the physical characteristics of the as spunfibres produced by a discontinuous method: Table 2 shows as spun fibreproduction rate and diameter data for PCL fibres formed from polymerhaving a mean molecular weight (MW) of 37,000. Table 2 shows that ingeneral fibres produced from polymers of lower molecular weight have areduced diameter.

In addition, alteration of the spinneret capillary orifice diameter wasfound to directly affect the diameter of as-spun fibres. Halving theorifice diameter from 1 mm to 0.5 mm resulted in a halving of thediameter of as-spun fibres produced from 10% w/v solutions. TABLE 2Production rates of as-spun PCL fibres. Spinning rates determined fordiscontinuous production of 200 mm lengths of fibre using MW 37,000fibre. PCL conc. Fibre production rate Fibre diameter (% w/v) (m/min)(μm) 5 fibres not formed 10 1.0 100 15 0.6 117 20 0.5 107 25 0.3 123Fibre tensile Properties

The effect of fibre spinning conditions on the tensile properties ofas-spun fibres is shown in Table 3. As-spun PCL fibres produced from 20%w/v solutions were found to exhibit a Young modulus of 0.1 GPa, UltimateTensile Strength (UTS) of 9.9 MPa and elongation at break ofapproximately 600%. Whereas, PCL fibres produced from 6% w/v solutionswhere found to have a Young modulus of 0.01 GPa, Ultimate TensileStrength (UTS) of 1.8 MPa and elongation at break of approximately 175%.This data shows that solution concentration can have a considerableeffect on physical characteristics. Variation in the physicalcharacteristics of the fibres may be useful in the production of suturematerials, variations in the strength of the fibres may make them usefulfor different textile applications. TABLE 3 The tensile properties ofas-spun PCL fibres Soln conc (% w/v) 6 10 15 20 Yield stress (MPa) 1.03.8 2.9 5.0 % extension at yield 17.1 10.8 15.9 8.7 Failure stress (MPa)1.8 7.9 6.1 9.9 % failure extension 175.4 514.9 429.0 596.1 E-modulus(GPa) 0.01 0.08 0.04 0.1

The effect of cold drawing on PCL fibre tensile properties is indicatedin Table 4. The general trend is an increase in tensile strength andstiffness and a decrease in failure extension with increasing draw ratioor extension. TABLE 4 The tensile properties of drawn PCL fibres Spunfrom 6% solution % extension 50 100 200 500 Yield stress (MPa) 6.5 8.415.0 N/A Failure stress (MPa) 12.3 13.7 20.0 N/A % failure extension260.1 188.2 107.6 N/A E-modulus (GPa) 0.06 0.09 0.1 N/A Spun from 10%solution % extension 50 100 200 500 Yield stress (MPa) 5.7 8.1 17.0 31.7Failure stress (MPa) 10.0 15.3 29.0 42.7 % failure extension 330.0 266.9369.2 140.5 E-modulus (GPa) 0.04 0.05 0.12 0.31 Spun from 15% solution %extension 50 100 200 500 Yield stress (MPa) 5.2 8.8 14.3 32.7 Failurestress (MPa) 8.2 16.3 21.7 47.0 % failure extension 213.5 260.3 302.8148.2 E-modulus (GPa) 0.05 0.11 0.11 0.24 Spun from 20% solution %extension 50 100 200 500 Yield stress (MPa) 8.0 13.0 22.3 29.3 Failurestress (MPa) 16.7 23.0 37.0 39.0 % failure extension 607.6 338.4 338.7136.3 E-modulus (GPa) 0.09 0.16 0.24 0.32Thermal Properties of PCL Fibres.

The melting point (Tm) and percentage crystallinity of as-spun PCLfibres produced from acetone solutions of various concentrations arepresented in Table 5. The Tm remained fairly consistent at around 56 °C. The crystallinity of the fibres tended to increase with increases inconcentration of the spinning solution. TABLE 5 The thermal propertiesof as-spun PCL fibres Solution conc. (% w/v) 6 10 15 20 Tm (° C.) 57.055.6 55.9 57.8 % crystallinity 63.7 66.2 74.1 75.3Surface Modification of PCL Fibres by Proteins.Gelatin Coating of PCL Fibres.

The amount of gelatin (used as a representative protein) adsorbed on thesurface of as spun 150 μm PCL fibres produced using a 10% (w/v) solutionof PCL in acetone following coating using various concentration proteinsolutions is shown in Table 6. The lower the gelatin solutionconcentration, the more gelatin was adsorbed. PCL fibres surfacemodified with extra cellular matrix proteins or cell adhesion ligandsmay be useful in modulating cell/fibre interaction and tissuedevelopment either in vivo or in vitro. TABLE 6 Gelatin adsorption onPCL fibres produced from a 10% w/v solution of PCL in acetone Conc. ofgelatin coating solution Wt gelatin/fibre surface area (% w/v) (μg/mm²)5 3.9 10 1.8 20 0.7OVA-Loaded PCL Fibres.

The amount of OVA exposed at the surface of OVA-loaded fibres (150 μmfibres produced using a 10% (w/v) solutioniof PCL in acetone) and theamount of OVA released into PBS at 37° C. increased with incubationtime, as shown in Table 7. These findings demonstrate a capacity forcontrolled presentation and release of proteins from the fibres. Thefibres may be used for the controlled delivery over time of an-array oftherapeutic agents such as those referred to earlier. TABLE 7 OVAexposure at the surface of protein-loaded fibres and release in PBS at37° C. Time in PBS Wt OVA/fibre surface area OVA release (days) (μg/mm²)of fibre (μg protein/mg of fibre) 1 1.4 3.4 2 4.8 6.0 7 9.3 15.8Progesterone-Loaded PCL Fibres

Steroid-loaded PCL fibres were produced by gravity spinning using a12.5% w/v solution of PCL in acetone containing 5% w/v of progesterone(4-pregnene-3,20 dione, Sigma Chemicals). Methanol was used as thenon-solvent and the fibre spinning rate was 2.1 m/min. As-spun fibreswere air dried for 2 days prior to testing. The release rate ofprogesterone from PCL fibres was investigated by incubating 35 mg offibres in 5 ml PBS at 37° C. The release medium was analysed atintervals for steroid content by measuring the absorbance at 248 nmusing a UV spectrophotometer and comparing with a calibration curve. Theamounts of progesterone release (μg/ml) were measured over 24 hours andare shown in Table 8 below.

The activity of released steroid was assessed by measuring its effect onbreast cancer cells in cell culture. Progesterone is known to retardgrowth of these cells. Breast cancer cells (MCF-7 breast epithelialcells) were seeded at a density of 2×10 4 in 24-well tissue cultureplates and allowed to attach and proliferate for 1 day. 30 mgs ofsteroid-loaded fibre were incubated in 5 ml of culture medium (DulbeccoMEM, ‘Invitron’) at 37° C. for 4 days. 0.5 ml aliquots of the releasemedium were added to each sample well and the cell growth rate wasmonitored over 3 days by cell counting (Neubauer haemocytometer,Appleton Woods). The cell proliferation rates over 3 days followingaddition of steroid released from PCL fibres are shown in FIG. 3together with control (TCP and PCL fibres with no steroid added).Inhibition of cell growth was observed demonstrating that the activityof progesterone was retained after fibre spinning and following in vitrorelease from the PCL fibres. TABLE 8 progesterone release from gravityspun PCL fibre Cumulative Progesterone Time Release Hrs μg 2 125.2 4210.9 8 341.1 14 463PCL Fibre/Cell Interaction.

Myoblasts were found to attach and grow in greater numbers initially(2-7 days) on as-spun PCL fibres (150 m fibres produced using a 10%(w/v) solution of PCL in acetone) relative to TCP controls (FIG. 4) andcell numbers per unit area were equivalent at day 8. The number offibroblasts attached to PCL fibres was higher than on TCP at day 4 (FIG.5). A large fall in cell number was measured at day 7 indicatingconfluence and contact inhibition. HUVECs showed (FIG. 6, average of 6replicates) comparable or greater attachment initially (1 to7 days),although there was subsequent growth (days 8 to 10) this was marginallyless than that achieved by the TCP control.

PCL fibres produced by the method of the invention proved to befavourable substrates for growth of fibroblasts and muscle cells. Thisproperty combined with the high fibre compliance recommends their usefor soft tissue reconstruction. The surface architecture of the fibrescan be modified by, for example, spinning onto a mandrel having specificsurface topography. This can enhance contact guidance of cells andimprove the fibres as substrates for cell growth.

Cell attachment and growth on biomaterials is known to be influenced bysurface physico-chemical properties (e.g. surface chemistry,hydrophobicity), microstructure (e.g. porosity) and the macrostructureor surface topography which gives rise to contact guidance effects.

Modification of PCL fibre surface topography was achieved by wrappingwet as-spun PCL fibres around a mandrel submerged in the methanol bathprior to the fibre drying stage. This facility enables the control offibre surface architecture or texture for modulating cell attachment andorientation via contact guidance effects. FIG. 7 illustrates how thesurface is modified (cf. FIG. 2).

Further potential uses for fibres producible by the methods of thepresent invention include:

-   -   Hard (e.g. bone) and soft tissue engineering (e.g. blood        vessels, muscle, nerves),    -   suture materials    -   textile vascular grafts, and    -   biodegradable fibres for textiles manufacture.

1. A method of producing a polymeric fibre comprising: (i) dissolving atleast one fibre forming polymer in a solvent so as to form a polymersolution, and (ii) feeding the polymer solution under gravity through anorifice directly into a non-solvent whereby to cause formation of apolymeric fibre in the non-solvent.
 2. The method as claimed in claim 1,wherein said at least one fibre-forming polymer is selected from alinear aliphatic polyester, a polylactide, a polyglycolide, theircopolymers with either (i) an aliphatic polyester, or (ii) polymersformed from monomers other than linear aliphatic esters.
 3. The methodas claimed in claim 2, wherein said polymer is selected frompoly(cεcaprolactone), poly(DL lactide co-glycolide) and poly(glycolideε-caprolactone).
 4. The method as claimed in claim 2, wherein thepolymer formed from monomers other than linear aliphatic esters isselected from at least one of poly(glycolide trimethylene carbonate),poly(L-lactic acid-L-lysine), poly(DL-lactide-urethane), PLA/PEOcopolymers, poly(ester-amide), a polyanhydride, a polyorthoester, apoly(ester-ether), a polyphosphazine, PHB, PHV and their copolymers, apoly(β-malic acid), a poly(amino acid), and an aliphatic or an aromaticpolycarbonate.
 5. A method of producing a poly(E-caprolactone) fibrecomprising: (i) dissolving poly(ε-caprolactone) polymer in a solventwhereby to form a poly(ε-caprolactone) solution, and (ii) feeding thepoly(ε-caprolactone) solution through an orifice directly into anon-solvent whereby to form said poly(ε-caprolactone) fibre.
 6. Themethod as claimed in claim 5, wherein when the at least onefibre-forming polymer is poly(ε-caprolactone), said solvent is selectedfrom one or more of acetone, ethyl acetate, dichloromethane,chloromethane and chloroform.
 7. The method as claimed in claim 6,wherein when the at least one fibre-forming polymer ispoly(ε-caprolactone) said non-solvent is selected from one or more ofmethanol, ethanol and water.
 8. The method as claimed in claim 1,wherein the non-solvent is chosen such that the polymer solution is moredense than the non-solvent.
 9. The method as claimed in claim 1, whereinstep (i) involves dissolving at least one additional polymer.
 10. Themethod as claimed in claim 1, wherein said at least one additionalpolymer is selected from poly(ε-caprolactone), PMMA, PU,poly(hydroxyethyl methacrylate), polyethylene glycol, polyethyleneoxide, copolymers of poly(ethyleneoxide)-poly(propylene oxide),polyvinyl pyrrolidone.
 11. The method as claimed in claim 1, whereinstep (ii) is effected by feeding the polymer solution through theorifice simultaneously with a pre-formed fibre, such that the resultantfibre comprises a core of the pre-formed fibre surrounded by a fibreformed from the polymer solution.
 12. The method as claimed in claim 11,wherein the pre-formed fibre is a polestar fibre.
 13. The method asclaimed in claim 1, wherein said solvent and non-solvent are miscible.14. The method as claimed in claim 1, including an additional step ofintroducing into the solvent one or more additives prior to step (ii).15. The method as claimed in claim 14, wherein said additional step iseffected by low shear mixing.
 16. The method as claimed in claim 1,comprising a step (iii) of applying at least one additive to the surfaceof the fibre formed in step (ii).
 17. The method as claimed in claims14, wherein the additive of said additional step and/or step (iii) isselected from one or more of a peptide, a protein, DNA, RNA,oligonucleotides, a polysaccharide including. inulin, starch, dextran,cellulose and derivatives, sugar spheres, extra-cellular matrixcomponents including glycosaminoglycans collagen, gelatin and albumin,bioceramics including hydroxyapatite, carbonate hydroxyapatite,tricalcium phosphate, carbon, calcium carbonate, and Bioglass, PMMApowders, polyesters, biodegradable polymers including PLA and PLG,polyorthoesters, polyanhydrides and oligosaccharide ester derivatives,discontinuous fibrous additives including alumina, carbon and syntheticpolymers including polyester, PGA and polydioxanone, polypeptide growthfactors including transforming growth factor-β, VEGF, EGF, BMP, IGF, orDNA encoding for polypeptide growth factors, their synthetic analoguesor conjugates with other molecules such as PEG, vaccine antigens,therapeutic antibodies, anti-coagulants, anti-cancer, anti-inflammatory,anti-bacterial and anti-viral agents, thrombolytic agents, hormones,decalcified freeze-dried bone and bioactive compounds for veterinary andagricultural use including pesticides, plant nutrients and growthhormones.
 18. The method as claimed in claims 1, wherein said additionalstep and/or step (iii) includes adding one or more types of celladhesion molecule.
 19. The method as claimed in claim 18, wherein saidcell adhesion molecules are selected from collagen, gelatin,fibronectin, vitronectin, laminin, elastin and their synthetic analoguesincluding protein-silk polymers or conjugates of such molecules withhydrophobic moieties, synthetic analogues of biomolecules containingcell binding sequences, antibodies having affinity for specific cellreceptors of interest and molecules having affinity for cell surfacepolysaccharides.
 20. The method as claimed in claims 1, wherein the oreach additive comprises one or more active agent and a carrier vehicle.21. The method as claimed in claim 20, wherein the carrier vehicle isselected from sugar spheres, spheres produced from polysaccharidesincluding dextran, inulin, starch, cellulose and derivatives, proteinsincluding gelatin and albumin, and synthetic carrier particles includingsynthetic non-resorbable polymeric particulates, microspheres andnanospheres, carbon, bioceramics and magnetic particles.
 22. A fibreproducible by the method of: (i) dissolving at least one fibre formingpolymer in a solvent so as to form a polymer solution, and (ii) feedingthe polymer solution under gravity through an orifice directly into anon-solvent whereby to cause formation of a polymeric fibre in thenon-solvent.
 23. A biomedical implant or cell-support matrixincorporating a fibre producible by the method of: (i) dissolving atleast one fibre forming polymer in a solvent so as to form a polymersolution, and (ii) feeding the polymer solution under gravity through anorifice directly into a non-solvent whereby to cause formation of apolymeric fibre in the non-solvent
 24. The method as claimed in claim 5,wherein the non-solvent is chosen such that the polymer solution is moredense than the non-solvent.
 25. The method as claimed in claim 5,wherein step (i) involves dissolving at least one additional polymer.26. The method as claimed in claim 5, wherein step (ii) is effected byfeeding the polymer solution through the orifice simultaneously with apre-formed fibre, such that the resultant fibre comprises a core of thepre-formed fibre surrounded by a fibre formed from the polymer solution.27. The method as claimed in claim 5, wherein said solvent andnon-solvent are miscible.
 28. The method as claimed in claim 5,including an additional step of introducing into the solvent one or moreadditives prior to step (ii).
 29. The method as claimed in claim 5,comprising a step (iii) of applying at least one additive to the surfaceof the fibre formed in step (ii).
 30. A poly(F-caprolactone) fibreproducible by the method of: (i) dissolving poly(p-caprolactone) polymerin a solvent whereby to form a poly(ε-caprolactone) solution, and (ii)feeding the poly(ε-caprolactone) solution through an orifice directlyinto a non-solvent whereby to form said poly(F-caprolactone) fibre. 31.A biomedical implant or cell-support matrix incorporating apoly(ε-caprolactone) fibre producible by the method of: (i) dissolvingpoly(ε-caprolactone) polymer in a solvent whereby to form apoly(ε-caprolactone) solution, and (iii) feeding thepoly(ε-caprolactone) solution through an orifice directly into anon-solvent whereby to form said poly(ε-caprolactone) fibre.